Structural characteristics of a neutral Glycyrrhiza uralensis polysaccharide and its fermentation properties on the gut microbiota of immunocompromised rats in vivo and vitro

Abstract

Glycyrrhiza polysaccharides (GPs) exhibit notable physiological activity; however, their structure is not well understood. The novel neutral polysaccharide fraction GP-1 was isolated from the roots and rhizomes of Glycyrrhiza uralensis Fisch. The fermentation properties of GP-1 were investigated in vitro and in vivo in immunocompromised rats. The molecular weight (Mw) of GP-1 is 7.6 kDa. The analysis of the monosaccharide composition indicated that GP-1 is a glucan. Nuclear magnetic resonance spectroscopy and methylation analyses revealed that GP-1 comprised a glucan main chain linked by α-D-Glcp-(1 → 4) bonds, with β-D-Glcp-(1 → 6)-α-D-Glcp-(1 → side branches at the C-6 position of 1,4,6-Glc and β-D-Glcp-(1 → side branches at the C-3 position of 1,3,4-Glc. Staining with Congo red confirmed the presence of a triple-helix structure. Scanning electron microscopy revealed that GP-1 has granular morphology. Pharmacological studies showed that GPs and GP-1 modulated the balance of the gut microbiota and influenced the production of short-chain fatty acids. In addition, the in vivo fermentation of GP and the in vitro fermentation of GP-1 promoted the growth of certain probiotics, particularly Lactobacillus and Dubosiella. These results underscore the structural characteristics of GP-1 and its potential as a prebiotic agent.

Keywords: Glycyrrhiza uralensis; fermentation; gut microbiota; neutral polysaccharide; structural characterisation.

Figure 1

Isolation and molecular weight determination of neutral polysaccharides in Glycyrrhiza uralensis (G. uralensis). (A) Extraction and purification scheme for GP-1. (B) Diethylaminoethyl (DEAE) elution profile. (C) High-performance gel permeation chromatography (HPGPC) results for GP-1.

Figure 2

High-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS) profiles of GP-1. (A) HPLC profile of monosaccharide standards. (B) HPLC profile following complete acid hydrolysis of GP-1. (C) GC–MS profile after methylation.

Figure 3

Infrared (IR) and nuclear magnetic resonance (NMR) spectra of GP-1. (A) IR spectrum of GP-1; (B) ¹H NMR spectrum of GP-1; (C) ¹³C NMR spectrum of GP-1; (D) Dept-135 NMR spectrum of GP-1; (E) HSQC spectrum of GP-1; (F) HMBC spectrum of GP-1; (G) COSY spectra of GP-1; (H) NOESY spectra of GP-1; (I) Structure of GP-1.

Figure 4

Scanning electron microscopy (SEM) and Congo red analyses of GP-1. (A) Maximum absorption wavelengths of Congo red-stained GP-1 at various NaOH concentrations. (B) SEM image of GP-1 (500 × and 20,000×).

Figure 5

Effects of glycyrrhiza polysaccharides (GPs) on immune function in immunocompromised rats (n = 6). A Effects on small intestinal villi. B: Effects on cytokine levels. CON: control group, MOD, model group; GP-L, low-dose GP group; GP-H, high-dose GP group. The values are expressed as means ± SD. The repeated measures data were analyzed using one-way ANOVA and Sidak post hoc test (vs. CON, *p < 0.05, ***p < 0.001; vs. MOD, # p < 0.05, ##p < 0.01, ###p < 0.001).

Figure 6

Overall gut microbiota composition in vivo and in vitro (n = 6). (A) Principal coordinate analysis (PCA) analysis at the operational taxonomic unit level for different groups. (B) Relative abundance at the phylum level; (C) Relative abundance at the genus level. CON, control group; MOD, model group; GP-L, low-dose GP group; GP-H, high-dose GP group; GP-1 L, low-dose GP-1 group; GP-1H, high-dose GP-1 group.

Figure 7

Linear discriminant analysis effect size (LEfSe) analysis of intestinal flora evolution in vivo and in vitro (n = 6). (A) Intestinal flora comparison between the in vivo CON and MOD groups; (B) Intestinal flora comparison between the in vivo MOD and GP-H groups; (C) Intestinal flora comparison between in vitro control and MOD groups. (D) Intestinal flora comparison between the in vitro MOD and GP-1H groups. CON, control group; MOD, model group; GP-H, high-dose GP group; GP-1H, high-dose GP-1 group (LDA > 4, p < 0.05).

Figure 8

Comparative analysis of representative intestinal bacteria (n = 6). (A) Representative intestinal flora in vivo. (B) Representative intestinal flora in vitro. CON, control group; MOD, model group; GP-L, low-dose GP group; GP-H, high-dose GP group; GP-1 L, low-dose GP-1 group; GP-1H, high-dose GP-1 group. The values are expressed as means ± SD. The repeated measures data were analyzed using one-way ANOVA and Sidak post hoc test (vs. CON, *p < 0.05, **p < 0.01, ***p < 0.001; vs. MOD, #p < 0.05, ##p < 0.01, ###p < 0.001).

Figure 9

Correlation heatmap of immunity and gut microbiota at the genus level (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 10

Levels of short-chain fatty acids (SCFAs) in vivo and in vitro (n = 6). (A) Representative chromatogram for SCFA quantification. (B) SCFA levels in vivo. (C) SCFA levels in vitro. CON: control group, MOD: model group, GP-L, low-dose GP group; GP-H, high-dose GP group; GP-1 L, low-dose GP-1 group; GP-1H, high-dose GP-1 group. The values are expressed as means ± SD. The repeated measures data were analyzed using one-way ANOVA and Sidak post hoc test (vs. CON, *p < 0.05, **p < 0.01, ***p < 0.001; vs. MOD, #p < 0.05, ##p < 0.01).